Ryanodine receptors (RyR) are sarcoplasmic reticulum Ca2+ release channels that play a critical role in Ca2+ signaling of excitable and non-excitable cells. RyRs owe their name to the fact that they were characterized in great part thanks to ryanodine, a plant alkaloid that binds to RyRs with high affinity and specificity. Ryanodine has been an invaluable ligand of RyRs, but its functional effects are complex and hamper its use in cellular studies. In search of novel ligands that could overcome some of the functional and structural disadvantages of ryanodine, we found in the venom of selected scorpions a set of peptide toxins, termed calcins, displaying high affinity and exquisite selectivity against RyRs. The defining characteristic of calcins is their capacity to stabilize RyR openings in a long-lasting subconducting state. This effect is nearly analogous to that of ryanodine, but unlike ryanodine, calcins bind rapidly to RyRs (fast association rate), freely dissociate from their binding site (reversible effect), display a dose- and sequence-variable effect, and are amenable for derivatization without undergoing major loss in receptor affinity. Calcins also modulate intracellular Ca2+ in intact cardiomyocytes with remarkable speed and with several degrees of potency, thus entering the field as the first cell-penetrating peptides (CPP) RyR-specific Ca2+ mobilizer of high dynamic range. This research program will characterize first and then exploit this novel group of peptide toxins to unravel fundamental mechanisms of RyR function at the molecular, cellular and whole heart level. Our multidisciplinary program, with well defined deliverables and milestones, may be enveloped in two specific aims. In the first aim, we will first identify and modify the structural domains of calcins involved in RyR recognition and cell penetration to generate a group of functionally diverse CPPs capable of modulating RyR function with wide dynamic range and of delivering cargo to the interior of cardiomyocytes. In the second aim, we will use calcins on native and recombinant RyR, in intact cardiomyocytes, and Langendorff-perfused working hearts to create acute or sustained periods of RyR hyperactivity and reveal mechanisms of RyR gating, Ca2+-triggered arrhythmias and electromechanical alternans. These studies will use ventricular cardiomyocytes as the cell model for characterization of calcins, but our ultimate goal is to generate for the scientific community a group of functionally diverse CPPs capable of modulating RyR function and of carrying cargo to the interior of a wide range of cells. PUBLIC HEALTH RELEVANCE: Ryanodine receptors are ubiquotous intracellular calcium channels that play a critical role in many aspects of cell function, including contraction, transcriptional regulation, enzyme activation, metabolic regulation, etc. but many aspects of their function still remain obscure. A major obstacle towards understanding the integral role of ryanodine receptors is the absence of specific ligands (molecules that bind and alter the activity of a molecule selectively and with high affinity). Recently, we discovered that selected scorpion venoms possess a group of peptide ligands that are able to penetrate cellular membranes and alter ryanodine receptor activity. In this study, we will exploit this novel group of peptide toxins to answer some of the most fundamental questions of ryanodine receptor structure and function. Results obtained here are therefore applicable to a wide range of cells.